Identification and development of specific monoclonal antibodies to squamous cell carcinoma

ABSTRACT

The present invention relates to novel antibodies, antibody fragments and antibody conjugates which display a high degree of selectivity for squamous cell carcinoma antigens including carcinomas of the lung, esophagus and cervix. The present invention relates to both in vivo and in vitro clinical screening methods for diagnosis or prognosis of carcinomas by means of detecting the expression of squamous cell carcinoma antigens in biological samples of the subject using the novel antibodies of the invention. The invention further provides for kits for carrying out the above described screening methods. Additionally, antibody conjugates may be used to efficiently deliver various agents which have anti-tumor effects to the tumor cell. The antibodies of the invention may also be administered to a patient in non-conjugated form to target ADCC to the tumor cell.

This application is a 371 of PCT/US01/26734 filed Aug. 28, 2001, whichclaims benefit of 60/229,785 filed Sep. 1, 2000, and claims benefit of60/230,890, filed Sep. 5, 2000.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Jan. 2, 2008. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as072771.0112.SEQLIST.TXT, is 1,464 bytes and was created on Dec. 29,2007. The Sequence Listing, electronically filed herewith, does notextend beyond the scope of the specification and thus does not containnew matter.

1. INTRODUCTION

The present invention relates to novel antibodies, antibody fragmentsand antibody conjugates which display a high degree of selectivity forsquamous cell carcinoma antigens including carcinomas of the lung,esophagus and cervix. The present invention relates to both in vivo andin vitro clinical screening methods for diagnosis or prognosis ofcarcinomas by means of detecting the expression of squamous cellcarcinoma antigens in biological samples of the subject using the novelantibodies of the invention. The invention further provides for kits forcarrying out the above described screening methods. Such kits can beused to screen subjects for expression of squamous cell carcinomaantigens as a diagnostic, predictive or prognostic indicator of cancer.Additionally, antibody conjugates may be used to efficiently delivervarious agents which have anti-tumor effects including, but not limitedto, chemotherapeutic drugs, toxins, immunological response modifiers,and radioisotopes, immunological response modifiers may also be providedin the form of fusion proteins. Immunological response modifiers mayalso be provided in the form of fusion proteins and radioisotopes mayalso be delivered while conjugated to single chain Fv chain constructs.The antibodies of the invention may also be administered to a patient inunconjugated form to target ADCC to the tumor cell.

2. BACKGROUND OF INVENTION

Lung cancer is one of the more common malignancy effecting thepopulation. Once a tumor is diagnosed, the overall prognosis is poor,with an overall five year survival rate of only 13%. However, earlydetection and treatment of lung cancer can significantly improve 5 yearsurvival rates. In those cases where the disease is detected early andsurgical resection is feasible, the five year survival rate increases to40%. For those at high risk for development of lung cancer intensivemonitoring has not been effective in reducing the incidence of thedisease or its outcome due in part to the inability to define earlystages of the disease when transformation is first occurring withinnormal cell populations of the tracheobronchial tree.

Monoclonal antibodies to tumor-associated antigens provide usefulreagents for diagnosis of cancer and for targeting of various anti-tumoragents such as radioisotopes, chemotherapeutic drugs and toxins to thesite of the tumor. Many monoclonal antibodies reactive againstcarcinoma-associated antigens are known. These known antibodies bind toa variety of different carcinoma-associated antigens that includeglycoproteins but for the most part bind to the carbohydrate moiety. Forexample, monoclonal antibodies that bind to glycoprotein antigens onspecific types of carcinomas include those described in U.S. Pat. Nos.4,737,579; 4,753,894; 4,579,827 and 4,713,352.

Since many tumor cells shed their membrane glycoproteins intosurrounding body fluids such as serum, or bronchial secretions in thecase of squamous cell carcinomas, the possibility of detecting shedantigen using monoclonal antibodies in an ELISA is a possible approachto early detection. This is the approach taken for detection of tumormarkers such as PSA and CEA in serum. As an example, supernatants of thebronchial lavage could be used in immunoassays designed for earlydetection of transformed cells in bronchial epithelium where shedantigen appears in the supernatant fluid. To date, assays designed todefine tumor cells in sputum samples have proven to be ineffectivebecause of the unavailability of monoclonal antibodies capable ofdifferentiating between normal and abnormal squamous cells or to be ableto define genotypically altered cells before the phenotypic changes ofmalignancy are evident.

Most studies have employed epithelial markers such as cytokeratinantibodies which are non-specific and will react with most if not allepithelial cells. Thus, better monoclonal antibody based diagnostic andprognostic markers and more sensitive tests for use in defining clinicallung cancer are needed. Ideally the monoclonal antibodies will detectthe expression of a specific tumor antigen at an early time whenclinical disease is not obvious. Such a tumor antigen defined by amonoclonal antibody could be used as a target for immunotherapy directedagainst the specific tumor antigen expressed on the surface of the cell.Such antibodies may also be of prognostic value based their ability toidentify markers expressed at different stages of disease or thatfunction differently, i.e., e-capherin, growth factor or receptor.

3. SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies, antibodyfragments, and antibody conjugates that are highly selective forsquamous cell carcinomas. More specifically, the novel antibodies,antibody fragments and antibody conjugates of the invention are thosethat bind to a cell membrane antigen found on squamous cell carcinomasbut show no or limited reactivity with normal cells or other types ofcancers. The invention also relates to hybridoma cell lines that producemonoclonal antibodies that are highly selective for squamous cellcarcinomas.

In yet another embodiment of the invention, the antibodies of theinvention may be used for in vitro or in vivo diagnostic and prognosticmethods designed to detect squamous cell carcinoma. For instance, theantibody may be used in methods designed to detect the presence of amalignant condition in human lung, cervical or other tissue. The tissuemay be contacted with an antibody of the invention which is capable ofdistinguishing squamous cell carcinoma cells from other cell types whichmay be present in the sample. Contact is carried out under conditionsthat allow for binding of the antibody to such cells followed bydetecting the presence or absence of binding of the antibody to thecells of the specimen. Additional diagnostic methods include the in vivolocalization of a tumor by administering to a patient a purifiedantibody or antibody fragment of the present invention labeled with anagent which gives a detectable signal. The localization of the tumor isthen detected using external scintography, emission tomography orradionuclear scanning.

The invention further relates to the use of the antibodies of theinvention in therapeutic applications, for example, to react withtargeted tumor cells. For example, novel antibody conjugates that act astarget selective carriers of various agents which have antitumor effectsincluding chemotherapeutic drugs, toxins, immunological responsemodifiers, enzymes and radioisotopes can be used.

Alternatively, the monoclonal antibodies may be used even in unmodified,i.e., not in conjugated form, to treat subjects having squamous cellcarcinoma. The antibodies of the present invention are particularly wellsuited for mediating antibody dependent cellular cytotoxicity (ADCC)which can results in targeted lysis of carcinoma cells in the presenceof human lymphocytes, macrophages and complement.

The invention also comprises the antigens identified by the antibodiesof the invention. Further encompassed are methods for using the purifiedor cloned antigens defined by the antibodies as vaccines to immunizeagainst certain squamous cell carcinomas.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Western Blot of Lung Lavage with Monoclonal Antibodies 5C6, AD6,AD7, and AH1.

FIG. 2. ADCC With Squamous Cell Carcinoma Specific Antibodies AH1 andAD7 and control antibody UPC-10.

FIG. 3A. ADCC With Squamous Cell Carcinoma Specific Antibodies AH1, AD6,AD7, 5C6 and control antibody UPC-10 against human squamous cellcarcinoma cell line H596. FIG. 3B. ADCC With Squamous Cell CarcinomaSpecific Antibodies AH1, AD6, AD7, 5C6 and control antibody UPC-10against human colon carcinoma cell line LS174T.

FIGS. 4A-F. Immunohistology Using Squamous Cell Carcinoma SpecificAntibodies AH6, AD7, and 5C6. FIG. 4A. Normal Lung Tissue probed withAD6. FIG. 4B. Normal Lung Tissue probed with AD7. FIG. 4C. Squamous CellCarcinoma Lung Lavage probed with AD6. FIG. 4D. Squamous Cell CarcinomaLung Lavage probed with 5C6. FIG. 4E. Squamous Cell Carcinoma probedwith 5C6. FIG. 4F. Squamous Cell Carcinoma probed with AD6.

FIG. 5. Direct ELISA assay with 5C6 and AD6 monoclonal antibodies usinglung lavage sample.

FIG. 6. Direct ELISA assay with 5C6, AD6, AH1 and AD7 monoclonalantibodies using lung lavage sample.

FIG. 7A. Pap Smear using 5C6 and AD7 monoclonal antibodies. Sliderepresents dysplasia but indicates the presence of cell expressingcarcinoma specific antigens.

FIG. 7B. In situ immunoperoxidase staining of cervical carcinoma cellsusing C56 (top) and AD7 (bottom).

FIGS. 8A, 8D 8E and 8H. LS174T colon carcinoma cells, H596 cells, H441cells and CaLu3 lung carcinoma cells stained with the AD7 antibody.

FIGS. 8B, 8C, 8F and 8G. LS174T colon carcinoma cells, H596 cells, H441cells and CaLu3 lung carcinoma cells stained with the AD6 antibody.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel monoclonal antibodies that arehighly specific for squamous cell carcinomas. More specifically, theantibodies react with squamous cell carcinoma of the lung and cervix,while showing none or limited reactivity with normal tissue. Suchantibodies include those designated AD6, 5C6, AD7, and AH1. All fourantibodies were reactive to lung cancer antigen and to human lung cancercell lines when assayed using ELIZA and immunofluorescent assays. Inaddition, the antibodies exhibit strong ADCC and immunohistochemicalactivity.

5.1 Squamous Cell Carcinoma Specific Antibodies

The present invention relates to novel antibodies that are highlyspecific for squamous cell carcinoma cells of the lung and cervix whileshowing none or limited reactivity with normal human tissue. The novelantibodies of the invention are designated AD6, 5C6, AD7, and AH1. Thedesignated antibodies can be used to isolate and characterize theantigen to which they bind. Thus, the antibodies can be used toidentify, isolate and/or characterize the immunogenic cell surfaceglycoproteins to which they react.

The term “AD6 antibody” as used herein includes whole, intact polyclonaland monoclonal antibodies such as the murine AD6 monoclonal antibodyproduced by hybridoma ATCC No. PTA-2460, and chimeric antibody moleculescapable of binding to the same antigenic determinant as the AD6antibody. The AD6 antibody described above includes fragments thereofcontaining the active antigen-binding region of the antibody, includingFab, F(ab′), and Fv fragments. The AD6 antibody of the invention alsoincludes fusion proteins.

The term “5C6 antibody” as used herein includes whole, intact polyclonaland monoclonal antibodies such as the murine 5C6 monoclonal antibodyproduced by hybridoma ATCC No. PTA-2458 (deposited on Sep. 6, 2000 withthe American Type Culture Collection at 10801 University Blvd., Mass.,VA 20110-2209 under the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure),and chimeric antibody molecules capable of binding to the same antigenicdeterminant as the 5C6 antibody. The 5C6 antibody described aboveincludes fragments thereof containing the active antigen-binding regionof the antibody, including Fab, F(ab′), and Fv fragments. The 5C6antibody of the invention also includes fusion proteins.

The term “AD7 antibody” as used herein includes whole, intact polyclonaland monoclonal antibodies such as the murine AD7 monoclonal antibodyproduced by hybridoma ATCC No. PTA-2459, and chimeric antibody moleculescapable of binding to the same antigenic determinant as the AD7antibody. The AD7 antibody described above includes fragments thereofcontaining the active antigen-binding region of the antibody, includingFab, F(ab′), and Fv fragments. The AD7 antibody of the invention alsoincludes fusion proteins.

The term “AH1 antibody” as used herein includes whole, intact polyclonaland monoclonal antibodies such as the murine AH1 monoclonal antibodyproduced by hybridoma ATCC No. PTA-2457, and chimeric antibody moleculescapable of binding to the same antigenic determinant as the AH1antibody. The AH1 antibody described above includes fragments thereofcontaining the active antigen-binding region of the antibody, includingFab, F(ab′), and Fv fragments. The AH1 antibody of the invention alsoincludes fusion proteins.

The present invention further encompasses the hybridoma cell linescapable of producing antibodies specific for squamous cell carcinomacell surface antigens. Such hybridoma cell lines include but are notlimited to hybridoma ATCC No. PTA-2457, hybridoma ATCC No. PTA-2459,hybridoma ATCC No. PTA-2458, and hybridoma ATCC No. PTA-2460.

Murine hybridomas which produce mAB specific for squamous cell carcinomacell surface antigens, such as the AD6, 5C6, AD7 and AH1 antibodies ofthe present invention, are formed by the fusion of a mouse fusionpartner cell, such as SP2/0 and spleen cells isolated from miceimmunized with squamous cell carcinoma cell surface antigens. Mice maybe immunized with crude or semi-purified preparations containing theantigens of interest. To immunize mice, a variety of differentconventional protocols may be followed. For example, mice may receiveprimary and boosting immunizations of antigenic preparations.

The monoclonal antibodies of the invention may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique of Kohler and Milstein, (1975, Nature256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,1983, Proc. NatL Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclasses thereof. The hybridoma producing the mAb of this inventionmay be cultivated in vitro or in vivo. Production of high titres of Mabsin vivo makes this the presently preferred method of production.

In addition, techniques developed for the production of “chimericantibodies” by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used(Morrison et al., 1984, Proc. Nat'l. Acad. Sci., 81:6851-6855; Neubergeret al., 1984, Nature, 312: 604-608; Takeda et al. 1985, Nature 314:452-454). Chimeric antibodies may be produced using a two-stephomologous recombination procedure such as that described in Fell etal., (1989, Proc. Nat'l. Acad. Sci., 86:8507-8511). Alternatively,techniques developed for the production of humanized antibodies (U.S.Pat. No. 5,585,089) or single chain antibodies (U.S. Pat. No. 4,946,778Bird, 1988, Science 242: 423-426; Huston et al., 1988, Proc. Nat'l.Acad. Sci USA, 85: 5879-5883; and Ward et al., 1989, Nature 334:544-546) may be used to produce antibodies that specifically recognizesquamous carcinoma.

The monoclonal antibodies of the invention may be produced in largequantities by injecting hybridoma cells secreting the antibody into theperitoneal cavity of mice and, after an appropriate time, harvesting theascites fluid which contains a high titre of the monoclonal antibody andisolating the antibody therefrom. The monoclonal antibodies may beproduced by culturing hybridoma cells in vitro and isolating thesecreted mAB from the cell culture medium (See, Cole et al., 1985, inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.).Alternatively, the gene for the antibody can be placed into a viralvector and used as a form of gene therapy so that the patient receivingthe antibody gene can produce therapeutic antibody through his ownimmune system.

In addition, antibody fragments including fragments thereof containingthe active antigen-binding region of the antibody may be prepared usingtechniques well established in the art (see, for example, 1986, Methodsin Enzymology 121:663-69(Academic Press)).

Also included within the scope of the invention are anti-idiotypicantibodies to the AD6, 5C6, AD7 and AH1 antibodies of the invention. Theanti-idiotypic antibodies can be produced using the AD6, 5C6, AD7 andAH1 antibodies and/or fragments thereof as immunogens. Suchanti-idiotypic antibodies are useful as diagnostic reagents fordetecting a humoral response to tumors and in therapeutic applicationssuch as in a vaccine, to induce an anti-tumor response in subjects withsquamous cell carcinomas.

Chimeric antibodies having the same binding specificity as the AD6, 5C6,AD7 and/or AH1 antibodies and combined with a cytotoxic agent areencompassed by the present invention. Such immunotoxins can be generatedusing genetic engineering techniques known in the art to producerecombinant DNA molecules capable of encoding a fusion proteincontaining the antigen binding region of the AD6, 5C6, AD7 or AH1monoclonal antibody fused to a cytotoxic agent such as, for example,diptheria toxin. In addition, humanized or completely human antibodiescan be produced using recombinant engineering techniques resulting inproduction of antibodies that induce less of an anti-mouse response.

The present invention encompasses novel antibody conjugates that act astarget selective carriers of various agents which have anti-tumoreffects including chemotherapeutic drugs, toxins, immunological responsemodifiers, enzymes and radioisotopes can be used. Such agents may beconjugated to the monoclonal antibodies of the invention for use intargeting the agent to the surface of squamous cell carcinoma cells.Such cytotoxic agents include for example vinca alkaloids, ricin, taxol,doxorubicin, methotrexate, mitomycin C, and cytochalasin B to name afew. Immunological response modifiers may also be provided in the formof fusion proteins and radioisotopes may also be delivered whileconjugated to single chain Fv chain constructs.

To generate the specific antibodies of the present invention,immunogenic glycoproteins derived from squamous cell carcinomas of thelung were isolated. The antigen was prepared from pooled allogeneicmaterial sampled at the time of surgery and fractionated by SephadexG-200 chromatography. Balb/c mice were immunized with the partiallypurified soluble membrane antigens of the human lung carcinoma cellsand, after a sufficient time, the mice were sacrificed and somaticantibody producing lymphocytes, e.g., spleen cells, were obtained andfused with the murine myeloma cell line SP2/0-Ag14.

Following fusion, the resulting cells were allowed to grow in selectivemedium, such as HAT-medium, and the surviving cells are grown in suchmedium using limiting dilution conditions and the supernatant wasscreened for monoclonal antibodies having the desired specificity.Various conventional methods exist for isolation and purification of themonoclonal antibodies, so as to free them from other proteins and othercontaminants.

Four hybrid clones were found to produce antibodies with the desiredspecificity. These antibodies were designated as AD6, 5C6, AD7, and AH1.All four monoclonal antibodies were found to reactive to lung cancerantigen and to human lung cancer cell lines as assayed by ELISA andimmunofluorescent assays. The antibodies did not react with normaltissue, bone marrow or tumor cell lines of other histological types. Inaddition, western blot analysis was performed using protein extractsderived from lung carcinoma cell lines P3 and P6 and partially purifiedlung cancer membrane antigens. Antigens of various molecular weight weredetected using the four different antibodies indicating that each of themonoclonal antibodies recognized a distinct antigen expressed on thecell membrane

These antibodies appear to identity biomarkers on an array of squamouscell cancers including lung, esophagus and cervix cancer. We havedemonstrated that the antibodies, i.e., 5C6 and AD7 are far moresensitive in cervical squamous malignancies than commercially availableantibodies. In addition, if the pap smear is performed using thin preptechnology so that cells are placed into fixative, the cytospin can beused as a source for measuring and defining a tumor antigen.

5.2. Diagnostic Assays for Detection of Squamous Cell Carcinoma SpecificAntigens

In accordance with the invention, the squamous cell carcinoma specificmonoclonal antibodies of the present invention can be used for the earlydiagnosis of diseases such as lung, esophagus and cervical carcinoma.Moreover, the monitoring and quantitation of antigen levels can be usedprognostically to stage the progression of the disease and to evaluatethe efficacy of agents used to treat a cancer subject. Antigen levelscan be monitored from cytospins by direct ELISA or in serum with acapture assay.

The detection of squamous cell carcinoma specific antigens in a samplefrom a subject can be accomplished by any of a number of methods.Preferred diagnostic methods for the detection of squamous cellcarcinoma specific antigens in the biological sample of a subject caninvolve, for example, immunoassays wherein the antigens are detected bytheir interaction with the specific antibodies of the invention.Antibodies useful in the present invention can be used to quantitativelyor qualitatively detect the presence of squamous cell carcinoma specificantigens or fragments thereof. For example, the monoclonal antibodies ofthe invention can be used, for example, to detect carcinoma cells inhistological and cytological specimens.

For instance, using an immunoperoxidase staining technique tissuespecimens can be analyzed for positive staining (Garrigues et al., 1982,Int. J.Cancer 29:511). Immunoassays useful in the practice of theinvention include but are not limited to assay systems using techniquessuch as Western blots, radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, to name but a few.

A biological sample which may contain squamous cell carcinoma specificantigens proteins, such as lung tissue or other biological tissue, isobtained from a subject suspected of having a particular cancer or riskfor cancer. Aliquots of whole tissues, or cells, are solubilized usingany one of a variety of solubilization cocktails known to those skilledin the art. For example, tissue can be solubilized by addition of lysisbuffer comprising (per liter) 8 M urea, 20 ml of Nonidet P-40surfactant, 20 ml of ampholytes (pH 3.5-10), 20 ml of 2-mecaptoethanol,and 0.2 mM of phenylmethylsulfonyl fluoride (PMSF) in distilleddeionized water.

Immunoassays for detecting expression of squamous cell carcinomaspecific antigens typically comprise contacting the biological sample,such as a tissue sample derived from a subject, with the monoclonalantibodies of the invention, i.e., AD6, 5C6, AD7, and AH1, underconditions such that an immunospecific antigen-antibody binding reactioncan occur, and detecting or measuring the amount of any immunospecificbinding by the antibody. In a specific aspect, such binding of antibody,for example, can be used to detect the presence and/or increasedproduction

A biological sample which may contain squamous cell carcinoma specificantigens proteins, such as lung tissue or other biological tissue, isobtained from a subject suspected of having a particular cancer or riskfor cancer. Aliquots of whole tissues, or cells, are solubilized usingany one of a variety of solubilization cocktails known to those skilledin the art. For example, tissue can be solubilized by addition of lysisbuffer comprising (per liter) 8 M urea, 20 ml of Nonidet P-40surfactant, 20 ml of ampholytes (pH 3.5-10), 20 ml of 2-mecaptoethanol,and 0.2 mM of phenylmethylsulfonyl fluoride (PMSF) in distilleddeionized water.

Immunoassays for detecting expression of squamous cell carcinomaspecific antigens typically comprise contacting the biological sample,such as a tissue sample derived from a subject, with the monoclonalantibodies of the invention, i.e., AD6, 5C6, AD7, and AH1, underconditions such that an immunospecific antigen-antibody binding reactioncan occur, and detecting or measuring the amount of any immunospecificbinding by the antibody. In a specific aspect, such binding of antibody,for example, can be used to detect the presence and/or increasedproduction of squamous cell carcinoma specific antigens wherein thedetection or increased production of squamous cell carcinoma specificantigens is an indication of a diseased condition. The levels ofsquamous cell carcinoma specific antigens in a biological sample arecompared to norms established for age and gender-matched normalindividuals and for subjects with a variety of non-cancerous orpre-cancerous disease states.

In an embodiment of the invention, the biological sample, such as atissue extract is brought in contact with a solid phase support orcarrier, such as nitrocellulose, for the purpose of immobilizing anyproteins present in the sample. The support is then washed with suitablebuffers followed by treatment with detectably labeled monoclonalantibodies such as AD6, 5C6, AD7, and/or AH1. The solid phase support isthen washed with the buffer a second time to remove unbound antibody.The amount of bound antibody on the solid support is then determinedaccording to well known methods. antibodies or antibody fragments, it ispossible to detect squamous cell carcinoma specific antigen expressionthrough the use of a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March 1986). Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

The monoclonal antibodies may also be labeled with a fluorescentcompound. Among the most commonly used fluorescent labeling compoundsare fluorescein isothiocyanate, rhodamine, phycoerythrin andfluorescamine. Likewise, a bioluminescent compound may be used to labelthe monoclonal antibodies. The presence of a bioluminescence protein isdetermined by detecting the presence of luminescence. Importantbioluminescence compounds for purposes of labeling are luciferin,luciferase and aequorin.

The antibodies of the invention can also be employed for in vivodiagnostic applications. For example, antibodies or fragments preparedfrom antibodies, can be used to image tumors, including metastatictumors in human patients. The purified antibody or fragments thereof arelabeled with an agent capable of giving a detectable signal andadministered in a suitable carrier, for example, intravenously, to apatient. The localization of the tumor-bound antibody is detected byexternal scintography, emission tomography or radionuclear scanning,using for example, a gamma camera.

5.3. Kits

The present invention further provides for kits for carrying out theabove-described assays. The assays described herein can be performed,for example, by utilizing pre-packaged diagnostic kits, comprising at anantibody reagent (for detection of squamous cell carcinoma specificantigens), which can be conveniently used, e.g., in clinical settings todiagnose disorders such as cancer. Such antibody reagents include themonoclonal antibodies AD6, 5C6, AD7, and AH1.

In a nonlimiting embodiment, a kit according to the invention maycomprise components which detect and/or measure squamous cell carcinomaspecific antigens in the biological sample of a subject. For example,where squamous cell carcinoma specific antigens are detected and/ormeasured by enzyme linked immunoabsorbent assay (ELISA), such componentsmay comprise an antibody directed to epitopes of the squamous cellcarcinoma specific antigens which can be used to detect and/orquantitate the level of squamous cell carcinoma specific antigensexpression in the biological sample. The antibody itself may bedetectably labeled with a radioactive, flourescent, calorimetric orenzyme label. Such antibodies include the monoclonal antibodies AD6,5C6, AD7, and AH1. Alternatively, the kit may contain a labeledsecondary antibody.

5.4 Therapeutic Uses of Monoclonal Antibodies

The antibodies of the present invention may be used therapeutically in avariety of different ways. The monoclonal antibodies may be used inunmodified, i.e., non-conjugated form, to treat subjects having squamouscell carcinoma. For example, the antibodies may be used to directcomplement (CDC) or effector cell (ADCC) mediated cytotoxicity.Alternatively, the antibodies may be conjugated to anti-tumor drugs,toxins or radionuclides. Conjugated antibodies can be administered topatients to achieve enhanced tumoricidal effects through the cytotoxicaction of the chemotherapeutic agent delivered to the tumor based on thebinding affinity of the antibody moiety.

Chimeric antibody molecules of the present invention may be preparedcontaining a mouse antigen-binding domain with human constant regiondomains (Morrison et al., 1984, Proc. Natl Acad. Sci. U.S.A. 81:6851;Takeda et al., 1985, Nature, 314:452) and this approach may be used togenerate novel antibody molecules with desirable effector functions suchas the ability to activate human complement and mediate ADCC.

The present invention relates to pharmaceutical compositions comprisingthe antibodies of the present invention. Amounts and regimens for theadministration of antibodies, their fragments or derivatives can bedetermined readily by those with ordinary skill in the clinical art oftreating cancer. For example, administration may be by parenteral,subcutaneous, intravenous, intramuscular, intraperitoneal, transdermalor buccal routes. Alternatively, or concurrently, administration may beby the oral route. The dosage administered will be dependent upon theage, health, and weight of the recipient, frequency of treatment and thenature of the effect desired.

Compositions within the scope of the invention include all compositionswherein the antibody, fragment or derivative is contained in an amounteffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges of effective amounts of each componentis within the skill of the art. The effective dose is a function of theindividual chimeric or monoclonal antibody, the presence and nature of aconjugated therapeutic agent, the patient and his clinical status andcan vary from about 10 ng/kg body weight to about 100 mg/kg body weight.The preferred dosages comprise 0.1 to 10 mg/kg body weight. Preparationsof the antibody, fragment or derivative of the present invention forparental administration, such as in detectably labeled form for imagingor in a free or conjugated form for therapy, include sterile lyophylizedprotein, aqueous or non-aqueous solutions, suspensions and emulsions.Examples of non-aqueous solvents are propyleneglygol,polyethyleneglycol, vegetable oil such as olive oil, and injectableorganic esters such as ethyloleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media, parenteral vehicles including sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, such as those based on Ringer's dextrose and the like.Preservatives and other additives may also be present, such as, forexample, anti-microbials, antioxidants, chelating agents, and inertgases and the like. See, generally, Remington's Pharmaceutical Science,16th ed., Mack Publishing Co., Easton, Pa., 1980.

In particular, the antibodies, fragments and derivatives thereof areuseful for treating a subject having a squamous cell carcinoma. Suchtreatment comprises parenterally administering a single or multipledoses of the antibody, fragment or derivative, or conjugate thereof.

The novel antigens of the invention to which the antibodies AD6, 5C6,AD7 and AH1 bind may also be used for therapeutic applications. Theantigens can be purified from tumors and administered, alone as animmunogen, or together with a proper immunological adjuvant.Alternatively, the antigens may be recombinantly produced for use as animmunogen.

6. EXAMPLE Production of Squamous Cell Carcinomaspecific MonoclonalAntibodies

The subsection below describes the generation of monoclonal antibodiesreactive against specific glycoproteins expressed on the surface ofsquamous cell carcinoma. Immunological assays indicate that themonoclonal antibodies react with carcinoma cells, while showing none orlimited reactivity with normal tissue. In addition AH1 and AD7.

6.1 Preparation of Monoclonal Antibodies

Monoclonal antibodies (mAb) against human squamous cell lung carcinomaassociated antigens (SLAA) were produced using a modified methoddescribed by Herzenberg et al. Four weeks old Balb/c mice were immunizedby intra peritoneal (ip) injection with 100 μg of partially purifiedSLAA obtained from the membrane extracts from pooled human squamous celllung carcinomas removed from patients at the time of surgery, emulsifiedwith 200 μl of complete Freund's adjuvant. Two ip injections emulsifiedin 200 μl of incomplete Freund's adjuvant were given at 2 weekintervals. Mice were then boosted intravenously with 20 μg of theimmunogen and the splenocytes were removed 3 days later. Somatic hybridswere prepared using the mouse non-secreting myeloma cell line SP2/0-Ag14following the procedue of Muraro et al. Hybridoma supernatents wereassayed for specific antibody production in ELISA using several humansquamous cell lung carcinoma cell lines (P3, P6 and H595) and partiallypurified immunogen. All hybridoma cell lines were cloned twice bylimited dilution.

For ascities fluids production, four week old Balb/c mice werepristane-primed and then inoculated with approximately 5×106 hybridomacells. mAb was purified from murine ascite fluids by protein G affinitychromatography. The protein content of purified antibody preparation wasanalyzed on a 12% SDS-polyacrylamide Tris-glycine gel.

6.2 Characterization of Monoclonal Antibodies

6.2.1. Western Blots

Western Blots were performed using the 5C6, AD6, AD7 and Ah1 antibodies.The protein samples were derived from lung lavage specimens. Samples (3μg) were loaded onto 12% SDS PAGE. Proteins were transferred tonitrocellulose membranes and stained using antibodies labeled withhorseradish peroxidase. As indicated in FIG. 1, the monoclonalantibodies of the invention each reacted against different antigensexpressed within squamous cell carcinomas derived from lung lavagespecimens.

6.2.2. ADCC with Squamous Cell Carcinoma Specific Antibodies

A four hour ⁵¹Cr release assay was used to measure ADCC activity. Targetcells were the lung squamous CA cell line H596. Target cells werelabeled with 200 μCi sodium [⁵¹Cr] chromate (250-500 mCi/mg, Amersham,Arlington, Ill.) in 200 μl fetal calf serum for 1 hour. Target cells(1×10⁴)were incubated in 96 U-bottom wells of assay plates with effectorcells in a ratio of effector to target cells of 80:1 in the presence ofmAb (1.0 μg/well). The plates were incubated fro 4 hrs at 37° C. in ahumidified atmosphere containing 5% CO2. Supernatant was harvested forgamma counting with the use of Skatron Harvester frames. Specific lysiswas calculated with the use of the following formula:

${{Specific}\mspace{14mu}{Lysis}\mspace{14mu}(\%)} = {\frac{{{Observed}\mspace{14mu}{release}\mspace{14mu}({cpm})} - {{spontaneous}\mspace{14mu}{release}\mspace{14mu}({cpm})}}{{{Total}\mspace{14mu}{release}\mspace{14mu}({cpm})} - {{spontaneous}\mspace{14mu}{release}\mspace{14mu}({cpm})}} \times 100}$

Spontaneous release was determined by measuring the radioactivityreleased from target cells incubated in medium alone. Total releasableradioactivity was obtained after treatment with 2.5% Triton X-100.Spontaneous release of radio-labeled chromium was measured aftertreatment with 2.5% Triton X-100.

As indicated in FIG. 2 and FIG. 3 the AH-1, D6 and 5C6 monoclonalantibodies demonstrated ADCC activity when tested against human squamouscell lung carcinoma cells. As presented in FIG. 3B, the ADCC activitywas specific for human squamous cell lung carcinoma cells as indicatedby the lack of activity against human colon carcinoma cells.

6.2.3. Immunohistochemistry Using Squamous Cell Carcinoma SpecificAntibodies

Staining protocols were as follows:

Mayo Frozen Tissue Protocol

Slides were fixed in 1% paraformaldehyde for 10 minutes followed byrinsing 3× in PBS. A blocking step was carried out with endogenousperoxidase (1% sodium azide +3% H₂O₂ and rinsed in water for 1 minute.5% NGS was added for 10 minutes followed by a addition of primaryantibody diluted in 1% NGS (2 μg/ml) and incubation for two hours at RT.Slides were rinsed 2-3× PBS and incubated for 15 minutes at RT withBiotin RAM (DAKO). The slides were rinsed 2-3× with PBS.Streptavidin(HRP) was added to the slides and incubated for 15 minutesat RT. The slides were rinsed twice with PBS and once with water. DAB orAEC.

Paraffin Section Protocol

Slides were placed at 60 degrees for 20 minutes. Deparaffinizing wasdone in the hood: (i) Xylene, 5 min; (ii) Xylene & 1% Iodine, 5 min:Xylene, 10 dips; (iii) 100% alcohol;(iv) 95% alcohol; and (v) 50%alcohol. Slides were placed in 50% methanol/50% 3% H₂O₂ for 10 min. andrinsed in water for 1 min. 5% NGS was added for 10 minutes followed byaddition of primary antibody diluted in 1% NGS (1 μg/ml) and incubationovernight at RT. Samples were rinsed in water and incubated with BiotinGAM (DAKO) for 30 min at RT. Slides were rinsed in water and incubatedwith Streptavidin (HRP) for 30 minutes (DAKO). Samples were rinsed andwater and DAB/AEC.

The four monoclonal antibodies of the invention were found to bereactive against antigens specifically expressed on the surface ofsquamous cell carcinomas of the lung (FIG. 4) but not on the surface ofnormal cells. In addition, the antibodies were found to beimmunoreactive against antigens expressed on the surface of cervicalcarcinoma cells (FIGS. 7A-B).

In addition ELISA assays were performed using the following protocol.Lung lavage was coated with dilution neat, 1:2, 1:4, 1:8, 1:16, 1:32,1:64, 1:128, 100 μl/well at 4° overnight. The wells were washed 3× withTBST. Blocking was carried out with TBST /2% gelatin (200 100 μl/well at37° for one hour followed by washing 5× with TBST. 100 μl/well ofascites antibody was added and incubated for 1 hr at 37°. Anti-mouse IgGAP 1:1000 was added and incubated for 1 hr at 37°. The wells were washed5× with TBST. Substrate P-NPP was added to each well and the endpointwas read at 405 nm. As indicated in FIGS. 5 and 6 the 5C6, and AD6monoclonal antibodies reacted with lung lavage tissue samples.

In addition, cell flow cytometry was performed to test the specificityof the antibodies. Cells (LS174T, H596, H441 and CaLu3) grown to logphase in culture medium free of phenyl red and removed from flasks with0.025% trypsin EDTA (BioWhittaker). The cells were then washed with PBS(pH7.4) and ssuspended in culture medium for 30 minutes and counted. Allsubsequent procedures were performed at 4° C.

The cells were washed three times with PBS. A sample containing 5×10⁵cells was suspended in 200 μl of PBS and delivered to each reactiontube. A solution of 200 μl of biotinylated antibody was added to thesamples and the mixture was incubated at 4° C. for 30 min. The cellswere washed three times with PBS and suspended in 500 μl of PBS. Thecells are then analyzed by flow cytometry. As indicated in FIG. 8, lungcarcinoma cells (H596, H441 and CaLu3) stained with the antibodies whilelittle staining was observed for the control colon carcinoma cells(LS174T).

6.3 Vaccine Studies

Membrane preparations were obtained from pooled allogeneic lung cancercells, i.e., squamous cell carcinomas. Membrane extracts were obtainedfrom viable cells which were then subjected to low frequency sonication.The soluble material was separated on Sephadex G-200 and furthersubjected to discontinuous polyacrylamide gel electrophoresis.Individual bands isolated from the gels and tested for DHR (delayedcutaneous hypersensitivity) as described above were pooled and used as avaccine. Skin testing for DHR employed 30 μg antigen (TAA). Sephadexfraction I was eliminated from the vaccine because this fractioncontained inhibitory material which suppressed the immune response. Thevaccine for therapy utilized 300 μg antigen in 0.2 ml complete FreundAdjuvant. In no instance was there evidence of an autoimmune pulmonaryresponse. In each patient an enhancement of cell mediated and humoralimmunity was observed. DHR continued to enhance over 5 years. 80% of thepatients studied (237 in study) survived after surgery and vaccinationversus 40% for surgery alone.

6.4 Cloning and Determination of the AD7 and 5C6 Heavy and Light Chains

The following primers are used for cloning and amplification of the AD7and 5C6 heavy chain V-region:

(i) cDNA: MHCGSP1A:

5′-CAT GGA GTT AGT TTG GGC AGC AGA-3′ (SEQ ID NO:1) Abridged AnchorPrimer;

5′-GGC CAC GCG TCG ACT AGT ACG GGIIGG GII GGG IIG-3′ (SEQ ID NO:2)

(ii) Amplification: MHCGSP2A:

5′-CAG GGG CCA GTG GAT AGA CAG ATG-3′ (SEQ ID NO:3)

Abridged Anchor Primer;

5′-GGC CAC GCG TCG ACT AGT ACG GGIIGG GII GGG IIG-3′ (SEQ ID NO:2)

The following primers are used for cloning and amplification of the AD7and 5C6 light chain V-region:

(i) cDNA: MLCGSP1:

5′-CCT GTT GAA GCT CTT GAC AAT GGG-3′ (SEQ ID NO:4)

Abridged Anchor Primer;

5′-GGC CAC GCG TCG ACT AGT ACG GGIIGG GII GGG IIG-3′ (SEQ ID NO:2)

(ii) amplification: MOSKAPPA:

5′-ACT GGA TGG TGG GAA GAT GGA T-3′ (SEQ ID NO:5)

Abridged Anchor Primer;

5′-GGC CAC GCG TCG ACT AGT ACG GGIIGG GII GGG IIG-3′ (SEQ ID NO:2)

6.5. Construction and Expression of Human-Mouse Chimeric Antibody Genes

Retroviral vectors pLHCXII and pLNCXII are used for the cDNA expressionof the chimeric heavy chain and light chains respectively. pLNCXII isthe vector LNCX except that an EcoRI site in the backbone of the vectoris destroyed while another EcoRI site located 45 basepairs 5′ to the neogene is retained. LNCX was obtained from Dr. D. Miller (Fred HutchinsonCancer Research Center, Seattle Wash.). The vector is derived from N2vector by replacing the 5′ long terminal repeats (LTR) of M-MuLV withM-MSV LTR and inserting a BalI/Xma III fragment containing the humancytomegalovirus (HCMV) immediate early promoter 3′ to the neo gene. Apolylinker containing HindIII, Hpa I and Cla I site is inserted 3′ tothe HCMV promotor for cloning of the target gene. The vector pLHCXII ismade by replacing a −1.2 KbEcoR1/BarnH1 fragment carrying the neomycinresistant gene with a 1.4 kb BamH1/BamH1 fragment carrying a geneconferring hygromycin resistance. For generating the retroviralexpression construct of pLNCXIIHuK, a DNA fragment carrying the cDNAencoding chimeric k chain (mAb) is obtained from its pBluescriptconstruct by cleavage with Sma I/Cla I. The resulting fragment is clonedin Hpa I/Cla I linearized pLNCXII vector. Similarly, the DNA fragmentsequences encoding the chimeric heavy chain is obtained from thepBluescript construct except that the plasmid is cleaved with Xba I, thesticky ends will be filled in by Klenow, and the fragment is excised bysubsequent cleavage with HindIII. The fragment generated is cloned inHindIII/Hpa I linearized pLHCXII to generate pLHXII HuG1, the expressionconstruct of chimeric mAb heavy chain.

For the development of chimeric mAb, the HC and LC expression constructsare sequentially introduced into SP2/O cells by electroporation, usingthe cell-porator system. Electroporation is carried out as follows.Briefly, SP2/O cells are washed in serum free DMEM with 4.5 g/L glucose(JRH Bioscience, Lenexa, Kans.) and suspended in the same medium at aconcentration of 5×10⁶ cells/ml. One hundred μg of the plasmid is addedto 1 ml of the cell suspension in a electroporating chamber at 4° C. Thecells and DNA mixture are pulsed at 650V/cm for 13 ms (capacitancesetting at 1,600 μF). Cells are kept at 4° C. for 10 min and diluted inRPMI1640 medium containing 15% FCS. Cells are then distributed in 24well plates at a concentration 5×10⁵ cell per well. After incubation at37° C. in 15% CO² incubator for 24 hour, selective medium containinghygromycin or active G418 at concentration of 500 μg/ml and 800 μg/mlrespectively is added.

Human mouse chimeric mAb (Hu mAb) producing clones are grown in proteinfree hybridoma medium PFHM-II (G1BCO,BRL) and are purified by protein Gaffinity chromatography. Protein concentration are determined usingBioRad microassay procedure or by method of Lowry. The protein isanalyzed on a pre-cast 4-20% SDS-polyacrylamide Tris-glycine gel (NovexSystem, San Diego, Calif.) with and without denaturation with2-mercaptoethanol. The protein gel is visualized by staining withCoomassie Brilliant Blue R250 according to the method of Lamemmli.

The present invention is not to be limited in scope by the specificembodiments described herein which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the claims. Variouspublications are cited herein, the contents of which are herebyincorporated, by reference, in their entireties.

1. A monoclonal antibody that binds to squamous cell carcinoma cellswherein the monoclonal antibody is mouse monoclonal antibody 5C6 asdeposited with the American Type Culture Collection and assignedaccession number PTA-2458.
 2. The antibody according to claim 1 which isdetectably labeled.
 3. The antibody according to claim 2 wherein saiddetectable label is a radiolabel.
 4. A fusion protein comprising theantigen binding region of monoclonal antibody 5C6, as deposited with theAmerican Type Culture Collection and assigned accession number PTA-2458.5. The fusion protein according to claim 4 which is detectably labeled.6. The fusion protein according to claim 5 wherein said detectable labelis a radiolabel.
 7. The fusion protein according to claim 4 which is ahuman-mouse chimeric antibody.